Comments

Comments on Primary Papers and News

Recent studies from a joint collaboration between our group and the Dietler laboratory at the EPFL provide, for the first time, direct evidence that Aβ42 fibrillization occurs via a combined mechanism of nucleated polymerization and secondary nucleation (see Jeong et al., 2013).

However, our AFM studies demonstrated that secondary nucleation occurs on the surface of only specific types of Aβ fibrils. For example, in the case of Aβ42, only "type 2" fibrils appear to be capable of growing via secondary nucleation events.

We did not investigate the generation of oligomers as a result of secondary nucleation events on the surface of fibrils, but the data provided by Knowles and colleagues provide strong evidence that this is possible.

It would be interesting to determine how the structural morphology of the parent fibrils influences the frequency and rate of secondary nucleation and what molecular factors and/or cellular events may influence this process.

More importantly, the results presented by Knowles and colleagues provide very nice mechanistic explanations for recent experimental observations from our group, suggesting that the process of nucleated polymerization plays a critical role in regulating Aβ toxicity. In this work, we showed that Aβ solutions containing a mixture of monomeric and aggregated Aβ are more toxic than solutions containing purely aggregated forms (fibrils or oligomers). We demonstrated a direct correlation between toxicity and the ability of Aβ species to form fibrils, and that this process depends on monomeric Aβ. Reintroduction of monomeric Aβ into nontoxic solutions containing Aβ protofibrils was sufficient to restore both toxicity and amyloid formation, whereas selective removal of monomeric Aβ from a toxic Aβ mixture (containing monomeric and aggregated Aβ species) was sufficient to block fibril formation.

We had postulated that fibrils associate with cellular membranes and that toxicity may be the result of 1) fibril-catalyzed formation of toxic Aβ species; 2) disruption of membranes as a result of the fibril formation process; or 3) activation of specific cellular pathways. The results reported by Knowles et al. provide evidence in support of the first, though we did not consider the possibility of secondary nucleation in this process (see Jan et al., 2011).

However, it is important to stress that Aβ toxicity may occur via multiple mechanisms involving the formation of specific Aβ aggregates or the process of Aβ fibril formation. There is no reason to think that there is a single toxic species or mechanism that is responsible for Aβ-induced neurodegeneration in AD.

This is a very nice study that brings together the use of several different approaches—mathematical modeling of protein polymerization, reliable assay systems to measure kinetics of Aβ aggregation, and innovative use of radiolabeled Aβ and size-exclusion chromatography. The conclusions reached build on a range of earlier findings including prior in-vitro data that also hinted at the importance of secondary nucleation (e.g., Wogulis et al., 2005; Jan et al., 2008; Jeong et al., 2013), and speculation that interaction between plaques (fibrils) and soluble Aβ is critical for toxicity (Koffie et al., 2009; Spires et al., 2009; McDonald et al., 2010; Esparaza et al., 2012). The most impressive aspects of this study are the quality of the kinetic data and the rigor of the analyses, both of which indicate that amyloid fibrils provide a catalytic surface for the generation of Aβ oligomers.

After thoughtfully considering the major factors believed to be important in Aβ aggregation (monomer concentration, nucleus formation, fibril elongation, and fibril fragmentation), the authors generated three predictive models and then tested them versus experimentally determined data. Thus, the outcome largely hinged on the use of a highly reproducible assay to monitor aggregation (Hellstrand et al., 2009). By examining the aggregation process under different shear conditions, they demonstrated that fragmentation of fibrils (as predicted) can also influence aggregation kinetics. Then, by seeding radiolabeled Aβ monomer with preformed unlabeled Aβ fibrils, they found that fibrils can catalyze the formation of oligomers, but that “ThT transparent” oligomers never account for more than 1 percent of the total mass of Aβ present. From Figure 4B, these oligomers appear to elute in or near to the void of a Superdex 75 column and therefore could be consistent with polydispersed prefibrillar assemblies such as protofibrils (Walsh et al., 1999; Harper et al., 1999) or ADDLs (Lambert et al., 1998; Hepler et al., 2006).

Finally, the authors present data suggesting that oligomers are biologically active. Although this is what one would expect, these experiments are the least solid part of the paper. Since it is not clear if these oligomers are stable during prolonged incubation and the toxicity assays were measured over 24 hours, it will be important to characterize oligomer activity using more rapid (and disease-relevant) assays. It will be similarly important to gain information about the structure of these oligomers and how they compare with assemblies isolated from human brain. Translating the lessons learned from this carefully controlled model to the chaos of the diseased brain will be challenging, not least because in the brain there are multiple Aβ peptides of different primary sequence and an abundance of surfaces on which to interact. Nonetheless, as an old-fashioned reductionist, I’m hopeful this study will help us inch a little closer to the worthy goal of preventing the formation (or neutralizing the effects) of toxic assemblies of Aβ.